Interactions between binding molecules, which in general are biomolecules, and their corresponding ligands, are central to life. Cells often bear or contain receptor molecules that interact or bind with a hormone, a peptide, a drug, an antigen, an effector molecule or with another receptor molecule. Enzymes bind with their substrate. Antibody molecules bind with an antigen, nucleic acid with protein, and so on. By “interact or bind” it is meant that the binding molecule and ligand approach each other within the range of molecular forces and may influence each others' properties. This approach takes the binding molecule and its ligand through various stages of molecular recognition comprising increasing degrees of intimacy and mutual effect: they bind, albeit not always irreversibly. Interactions between binding molecules are widely and extensively tested in the field of candidate drug testing, with the ultimate goal to find specific drugs that can interact or bind with specific target molecules in the body that mediate or modulate the development of disease.
Binding molecules have binding ability because they comprise distinct binding sites allowing for the recognition of the ligand in question. The ligand, in turn, has a corresponding binding site and only when the two binding sites can interact by essentially spatial complementarity, the two molecules can bind. Needless to say that, molecules having three dimensions, binding sites are of a three-dimensional nature, often one or more surface projections or protuberances of one binding site correspond to one or more pockets or depressions in the other, a three-dimensional lock-and-key arrangement, sometimes in an induced-fit variety. Sometimes, such a protuberance comprises a single loop of the molecule in question and it is only this protuberance that essentially forms the binding site. In that case, one often terms these binding sites as comprising a linear or continuous binding site, wherein a mere linear part of the molecule in question is in essence responsible for the binding interaction. This terminology is widely used to describe, for example, antibody-antigen reactions wherein the antigen comprises part of a protein sequence, a linear peptide. One then often speaks about a linear or continuous epitope, wherein the binding site (epitope) of the antigenic molecule is formed by a loop of consecutively bound amino acids. However, similar continuous binding sites (herein, “epitope” and “binding site” are use interchangeably) can be found with receptor-antigen interactions (such as with a T-cell receptor), with receptor-ligand interactions such as with hormone receptors and agonists or antagonists thereof, with receptor-cytokine interactions or with for example enzyme-substrate or receptor-drug interactions, wherein a linear part of the molecule is recognized as the binding site, and so on. More often, however, such a protuberance or protuberances and depressions comprise various, distinct parts of the molecule in question, and it is the combined parts that essentially form the binding site. Commonly, one names such a binding site comprising distinct parts of the molecule in question a discontinuous or conformational binding site or epitope. For example, binding sites laying on proteins having not only a primary structure (the amino acid sequence of the protein molecule), but also secondary and tertiary structure (the folding of the molecule into alpha-helices or beta-sheets and its overall shape), and sometimes even quaternary structure (the interaction with other protein molecules), may comprise in their essential protuberances or depressions amino acids or short peptide sequences that lay far apart in the primary structure but are folded closely together in the binding site. In linear (continuous) binding sites, the key amino acids mediating the contacts with the antibody are typically located within one part of the primary structure usually not greater than 15 amino acids in length. Peptides covering these sequences have affinities to the target proteins that are roughly within the range shown by the intact protein ligand.
In conformational (discontinuous) binding sites, the key residues are in general distributed over two or more binding regions, which are often separated in the primary structure. Upon folding, these binding regions can be brought together on the protein surface to form a composite binding site. Even if the complete binding site mediates a high affinity interaction, peptides covering only one binding region, as synthesized in a linear scan of overlapping peptides, generally have very low affinities that often cannot be measured, for example, in normal ELISA or Biacore experiments.
The discovery of the physiological role of a great number of peptides stimulated researchers all over the world towards design and synthesis of peptidomimetics (or peptide-like molecules) as candidate drugs or diagnostic tools. Since natural peptides seldom can be used therapeutically as drugs because of the problems associated with low absorption, rapid metabolism and low oral bioavailability, many efforts aimed to modify the natural sequence of the amino acids of bioactive peptides achieved a desired, very focused effect. Modern biochemical techniques have identified a large number of peptides having potent pharmacological activities. However, since peptides are not usually orally active and suffer from short half lives in vivo, their direct utilization as drugs is not generally feasible. In addition, the interactions of many peptides with their macromolecular targets (receptors, enzymes, antibodies) will depend on the adoption of a particular conformation. Accordingly, the design of conformationally restricted peptides and the partial replacement of peptides with bioisosteric units mimicking these peptide binding sites have become contemporary goals of medicinal chemistry. Synthetic non-natural peptides (or pseudopeptides or peptidomimetics) have the advantage of providing new functionalities that can circumvent natural processes in the body. For example, they become able to perform functions that are not available with the natural materials, such as binding to and penetrating cell membranes and resisting degradation by enzymes.
Candidate drug testing is these days often performed on a high-throughput scale, wherein arrays of candidate compounds, such as libraries of peptides or nucleic acid molecules attached to solid supports are contacted with target molecules that are thought to be relevant to one or more aspects of a disease under study. Binding of such a target molecule to such a candidate compound is then seen as a possible hit or lead towards the identification of a binding site of the target molecule and, simultaneously, towards the identification of a candidate compound, from among the many different compounds present on the array, having a binding site that, more or less, bears relevance for interaction with the target molecule. However, having identified a lead compound in no way means that one has selected a definite drug compound suitable for interaction with the target molecule. For one, the binding site identified may only partly fit or be relevant for the molecule in question, and several rounds of modification may be required before a better fit, and thus a more appropriate binding site, has been identified. Also, considering that all the testing so far has been done in an array format, or at least with molecules attached to a solid support only, no attention has yet been given to the fact that a drug needs to be administered in solution, away from the solid support on which its lead was identified. As molecules often change or behave quite differently in solutions, having lost the specific constraints when attached to a solid phase, many promising lead compounds actually lose their attraction as a candidate drug when tested for the interaction with their target molecule in solution, again necessitating various rounds of modification before their candidacy as a drug may become further established.
Mimicking binding sites of complex proteins, e.g. TNF-alpha, the CD (cluster of differentiation antigen)-family, cytokines, or protein binding sites, like antibodies or cell surface receptors, by means of synthetic peptides or equivalent bioisosteric units is currently one of the most active areas in protein science and drug development. Many proteins exert their biological activity through interactions involving relatively small regions of their exposed surfaces. Molecules that mimic these surface epitopes are, therefore, of great interest, since they may provide a means of mimicking the biological activity of the entire protein in a relatively small synthetic molecule. Short linear peptides are not ideal for this purpose because of their inherent flexibility and susceptibility to proteolytic degradation. Instead, it is preferred to constrain linear peptide chains by cyclization into biologically relevant secondary structures. Thus, the challenge for the development of successful binders is primarily related to fixing the essential peptide sequence in the correct conformation and orientation on a platform or scaffold. The conformational rigidification of a single linear peptide, like backbone or side-chain cyclization strategies, has given rise to numerous cyclopeptides with subnanomolar activities. Various procedures to obtain such monocyclic peptides are fairly well worked out and procedures for their synthesis are also available. Various efficient synthetic routes to scaffolds for preparing monocyclic peptides have been developed, along with methods for their incorporation into peptidomimetics using solid-phase peptide synthesis. Among the various approaches utilized for preparing monocyclic peptides, several employed peptides containing pairs of cysteine residues allowing subsequent cyclization via disulfide bond formation (see, for example, U.S. Pat. No. 3,929,758; U.S. Pat. No. 4,518,711; U.S. Pat. No. 5,216,124; U.S. Pat. No. 5,169,833; WO9109051; and WO9108759).
In sharp contrast, the synthesis of scaffold-bound peptidomimetics with multiple peptides or peptide segments, for example, for mimicking discontinuous epitopes or binding sites, has been facing long-standing technical difficulties. Not only multiple peptides or peptide segments need to be fixed on a molecular scaffold or scaffolds, but typically they have to be captured in a structurally coordinated fashion to be effective as a binding partner. Numerous efforts have been devoted to the synthesis of conformationally constrained peptide constructs consisting of multiple looped peptide segments. However, only a few examples exist in which the conformational fixation of multiple constraint peptide loops on a (synthetic) platform is achieved. A major problem is that suitable chemistry is far from straightforward. Current approaches essentially always require multiple protection/deprotection schemes in order to couple more than one different peptide or peptide segment onto a functionalized scaffold molecule in a controlled fashion. A functionalized scaffold molecule refers to a molecule serving as a scaffold or scaffold for another molecule wherein the scaffold is provided with multiple, usually different, functional groups via which the other molecules can be attached. For example, binding of a peptide via its N-terminal amino group to a functionalized scaffold or scaffold requires protection of all amino acid side chains also containing a reactive amino group, like lysine or arginine residues, in order to prevent unwanted coupling of such a side chain to a scaffold. Likewise, acidic amino acids need to be protected when using a coupling procedure via the C-terminal carboxyl group of a synthetic peptide. Following completion of a product, further deprotection or cleavage steps have to be performed because the side chain protection groups must be removed to recover the original amino acids. Frequently, depending on the nature of a protective group, the removal of each type of protective group requires a different protocol involving, for example, different buffers, solvents and chemicals. As a consequence, a long and tedious course of action is required to be able to isolate the desired product in measurable quantities when using procedures available thus far. Furthermore, an additional problem when working with purely synthetic scaffolds is the required selective functionalization, which ultimately leads to multistep procedures, often with disappointingly low yields.